Relationship Between Phosphorylation-related Genetic Variants And The Risk Of Colorectal Cancer

Colorectal cancer (CRC) is the fourth common malignancies. In China, it is estimated that more than250,000new cases occur every year, and the incidence is rising these years, suggesting that there is no time to delay for CRC prevention. Extensive epidemiological studies and molecular experiments have indicated that CRC is a complex trait, caused by long-term effect of genetic predisposition and environmental exposures. Several environmental risk factors have been reported to be associated with CRC risk, such as cigarette, alcohol, high-fat diet and insufficient physical activities. However, only a few exposed individuals ultimately develop CRC, implying that genetic components also have an important role in CRC risk. Nowadays along with the rapid progress of genomics, there are many CRC susceptible loci identified by genome-wide association studies (GWAS) and candidate gene researches, but these findings are still not enough to fill in the blank of understanding of genetic mechanism of CRC. Hence it is warranted to dig into more potentially functional loci from multiple viewpoints, aiming to construct the CRC’s genetic architecture.Protein phosphorylation is a very important post-translational modification that takes part in almost every process of cellular activities. The phosphorylation-regulating system is composed of protein substrates, kinases that catalyze the transfer of phosphate groups to specific substrates, and phosphatases that catalyze the remove of phosphate groups from phosphorylated substrates. Abnormal phosphorylation results in aberrant activities of substrates and/or their abnormal interaction with other molecules, thus affecting cell’s life including growth, proliferation, differentiation and apoptosis, finally involving the pathogenesis of human diseases. Increasing numbers of phosphorylation-related variants have been identified to be associated with various cancers via integrating data of genomics and phosphorylated proteomics. Moreover, genetic polymorphisms occur in kinases, phosphatases and substrates have been reported to modify the risk of cancers. Up till now, the role of kinases in tumorgenesis is widely accepted based on accumulated evidences. However, the potential mechanism of aberrant phosphatases/substrates in tumors is still stuck on the discovery stage, requiring more and deeper investigations including population studies and functional assays. Therefore, from the perspective of phosphatases and substrates, we used bioinformatic analysis and case-control studies, followed by molecular functional assays, to investigate the association between phosphorylation-related genetic variants and the CRC risk.Part I A Phosphorylation-related Variant ADD1-rs4963Modifies the Risk of Colorectal CancerBackground:ADD1(a-adducin) is an important component of membrane cytoskeleton, and plays a crucial role in cell proliferation, motility and communication. Previous researches have indicated that ADD1has a potential effect on tumorgenesis. Recently, a missense variant at the codon of ADD1’s phosphorylation site, rs4963(Ser586Cys), was reported to modify the risk of non-cardia gastric cancer.Objectives:To investigate the association of ADD1-rs4963with CRC risk in Chinese population; to explore the potential effect of gene-environment interaction on the susceptibility to CRC.Methods:A hospital-based case-control research was performed in1054CRC cases and1128matched controls to explore the relationship of ADD1-rs4963with CRC risk, using by TaqMan genotyping technology. Differences between cases and controls were evaluated by Pearson’s χ2or the independent-samples t test in the distribution of demographic characteristics, including age, gender, smoking and drinking status. The association of ADDl-rs4963with CRC risk was estimated with the odds ratio (OR) and the corresponding95%confidence interval (95%CI), via unconditional logistic regression analysis without or with adjustment for gender, age, smoking and drinking. The potential gene-environment interaction between ADD1-rs4963and smoking or drinking was assessed by the genotype-smoking or genotype-drinking combined effect and the multiplicative interaction term in logistic model.Results:The allele frequency differed significantly between cases and controls (P-0.011). A similar result was also shown for the genotype distribution (P=0.028). After adjustment for age, gender, smoking and drinking, an increased risk of CRC was statistically associated with CG (OR=1.25,95%CI=1.02-1.55, P=0.036) and GG genotype (OR=1.35,95%CI=1.06-1.72, P=0.015) compared with CC genotype. In the dominant model, CG and GG individuals showed a1.28-fold higher risk than CC individuals based on multifactorial logistic analysis (OR=1.28,95%CI=1.05-1.57, P-0.014). Moreover, the G allele demonstrated more susceptibility to CRC, with an adjusted OR of1.16(95%CI=1.03-1.31, P=0.016) under the additive model. All the evidences strongly suggested that this variant was significantly associated with CRC risk. In addition, we performed interaction analyses between ADD1-rs4963and smoking or drinking exposure, but found no significant result.Conclusion:ADD1-rs4963significantly conferred susceptibility to CRC in Chinese population. This study is the first report of an association between ADD1and CRC risk, promoting our knowledge of the genetics of CRC.Innovations:Our work firstly explored the relationship of ADD1with susceptibility to CRC in Chinese population, partly filling the blank of domestic association studies between phosphorylation-related genetic variants and cancer risk. Part Ⅱ Association between Missense Polymorphisms of Classical Protein Tyrosine Phosphatases and the Risk of Colorectal CancerBackground:Protein tyrosine phosphatases (PTPs) can counter the activities of tyrosine kinases and keep equilibrium of cellular phosphorylation, thus playing a crucial role in regulating cell growth, proliferation, apoptosis and motility. As an earliest found and most studied subfamily, classical PTPs have been reported to be associated with various human cancers.Objectives:To systematically evaluate the association between missense polymorphisms of classical PTPs genes and CRC risk in Chinese population; to deeply analyze the potential gene-environment interactions between candidate loci and smoking or drinking exposure; to further explore the molecular functions of significant loci identified by our two-stage association study, and investigate the potential mechanism in tumorgenesis of CRC.Methods:Firstly, we systematically identified missense polymorphisms of classical PTPs genes by the bioinformatic analysis. Secondly, a two-stage case-control strategy was performed to investigate the association between candidate loci and CRC risk. We used unconditional logistic regression analysis to assess the adjusted effect sizes of candidate loci, after the adjustment for common confounding factors such as age, gender, smoking and drinking. The potential gene-environment interactions were also analyzed under the logistic model. At last, we carried out protein function assays to further explore the potential molecular effect of positive loci on the CRC pathogenesis.Results:A total of25candidate loci were selected by bioinformatic screening. At the discovery stage, we recruited1096CRC cases and1816matched controls and identified two significant loci; one is rs3750050at PTPN12gene (P-ajusud=2.220×10-4, FDR-adjusted=0.004), another is rs6780013at PTPN23gene(P-ajusted=3.645×10-4, FDR-adjusted=0.003). At the validation stage, we performed an association study including767CRC cases and1215matched controls, and successfully validated the significant polymorphism PTPN12-rs3750050. Results from the combined analysis of two-stage data demonstrated that rs3750050-G was a risk allele for CRC. Specially, for each G allele carried in an individual, a25%increase was found in the risk of CRC (OR=1.25,95%CI=1.14-1.36, P=8.158×l0-7). Further analysis of gene-environment interaction showed that there was significant interaction between PTPN12-rs3750050and smoking(P吸烟=0.013). Individuals exposed with both GG risk genotype and smoking would be more susceptible to CRC (OR=1.50,95%CI=1.24-1.83). Then we carried out a western blot assay to explore the potential effect of this variant and found that G allele weakened PTPN12’s dephosphorylation at Tyr416of Src, which is an important substrate that regulates cell growth and proliferation in intestine cell. The reduced dephosphorylation would result in abnormal activation of Src, thus possibly making effects on the pathogenesis of CRC.Conclusion:1. PTPN12-rs3750050was shown a significant association with CRC risk. Specially, rs3750050-G could confer risk to CRC in Chinese population.2. There was an obvious interaction between PTPN12-rs3750050and smoking. Cigarette exposure further increased the susceptibility to CRC for individuals with GG risk genotype.3. PTPN12-rs3750050influenced the phosphatase activity of PTPN12, and G risk allele weakened PTPN12’s dephosphorylation on Src, thus possibly playing a role in tumorigenesis.Innovations:Our work was the first to systematically evaluate the association between missense polymorphisms of classical PTPs genes and CRC risk in Chinese population by using bioinformatics, two-stage association study and functional assay. Moreover, gene-environmental interaction analyses were applied to explore potential interactions of candidate loci with smoking or drinking exposure. At last, we provided some functional evidence to support our identified association via the western blot assay.